Recognition of noncanonical poly(A) sites in human genes

Cleavage and polyadenylation are essential pre-mRNA maturation reactions for almost all eukaryotic mRNAs. Noncanonical poly(A) sites are frequently present at proximal sites during alternative cleavage and polyadenylation. These sites are involved in the regulation of gene expression. This project aims to elucidate the mechanism of noncanonical polyadenylation. Two noncanonical poly(A) sites JUND and ARAF have been investigated. Analysis of the sequence requirement of the AGUAAA poly(A) site in JunD reveals that, besides the core sequence element, a U-rich element (adU-rich) is required for optimal usage and the distance between AGUAAA and this element is important; Similar to MC1R, the ARAF poly(A) site has a degenerated poly(A) signal and it has been suggested to require a distant auxiliary element, possibly G-rich enhancer, for optimal poly(A) site usage. With regard to trans-acting factors, WDR33 appears to be important for the recognition of sub-optimal JunD poly(A) sites, whereas CPSF30 and CPSF160 preferentially recognise the canonical poly(A) site over a weak poly(A) site in JunD-MC4R 3' end formation. Contrary to the current models, depletion of CFIm68 does not favour proximal noncanonical poly(A) site usage in our JunD-MC4R Mini-gene system, but instead reduces the usage of the distal site and generates read through transcripts. Finally, we find that whilst WDR33 and CPSF30 affect poly(A) site selection in JunD-MC4R plasmids, they globally do not show general preference for either canonical or noncanonical sites. In summary, we investigate the architecture of ARAF and JunD poly(A) sites. We conclude that in both cases the strength of the poly(A) site is determined by the complex architecture of the poly(A) sites rather than the core sequences. Recognising the wider architecture of poly(A) sites will be essential to fully understand what constitutes a strong poly(A) site and how this can be modulated, in particular in the context of alternative cleavage and polyadenylation.